P-Glu-D-Phe-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Gly-NH{HD 2 {B and intermediates

ABSTRACT

D-Phe2-D-Leu6-LRF, is described as well as its synthesis by solid phase techniques and novel intermediates formed by such synthesis. The novel decapeptide exhibits anti-ovulatory activity in mammals.

United States Patent 11 1 Yardley [451 May 27, 1975 1P-GLU-D-PHE-TRP-SER-TYR-D-LEU-LEU- ARG-PRO-GLY-NH AND INTERMEDIATES [75]Inventor: John P. Yardley, King of Prussia,

[73] Assignee: American Home Products Corporation, New York, NY.

[22] Filed: Nov. 21, 1973 [21] Appl. No.: 417,983

[52] US. Cl 260/1125; 424/177 [51] Int. CL. C070 103/52; CO7g 7/00; A6lk27/00 [58] Field of Search 260/1 12.5

[56] References Cited OTHER PUBLICATIONS Fujino et aL: Biochem. Biophys.Res. Comm., 57, 12481256 (1974).

Primary Examiner-Lewis Gotts Assistant Examiner-Reginald J. Suyat 8Claims, No Drawings P-GLU-D-PHE-TRP-SER-TYR-D-LEU-LEU-ARG- PRO-GLY-NHAND INTERMEDIATES This invention relates to the novel decapeptide p-Glu-D-Phe-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Gly-NH its process ofmanufacture and novel intermediates formed in such synthesis.

The luteinizing hormone releasing factor (hereafter called LRF) is thedecapeptide, L-(5-oxoprolyl)-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L-arginyl-L-arginyl-L-prolyl-glycine amide. This decapeptide is secretedby the hypothalamus and carried to the adenohypophysis where itstimulates the release of the luteinizing hormone and the folliclestimulating hormone. The present invention concerns itself withstructural modifications of LRF which exhibit antiovulatory activity.

The novel peptides of the present invention are represented by thecompounds of the formula:

p-Glu-D-Phe-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Glyand its non-toxic salts.All chiral amino acid residues identified in formula I supra, and theother formulas hereinafter are of the natural or L-configuration unlessspecified otherwise.

Also contemplated within the scope of the present invention areintermediates of the formula wherein:

R is selected from the class consisting of NH OH, O-(lower)alkyl, inwhich (lower)alkyl is C through C (e.g. methyl, ethyl, pentyl, hexyl,etc.) and O-benzyl;

N means the side chain nitrogen atoms of arginine;

R is at protecting group for the N N and N nitrogen atoms of arginineselected from the class consisting of nitro, tosyl, benzyloxycarbonyl,and adamantyloxycarbonyl; or R is hydrogen which means there are noprotecting groups on the side chain nitrogen atoms of arginine. Wherethe protecting group is nitro or tosyl, the protection is on either oneof the N N"" nitrogens and in the case of benzyloxycarbonyl, oradamantyloxycarbonyl, the protection is on the N nitrogen and either oneof the N N nitrogen atoms. The preferred protecting group defined by Ris nitro;

R is a protecting group for the phenolic hydroxy] group of tyrosineselected from the class consisting of acetyl, tosyl, benzoyl,tetrahydropyranyl, tert-butyl, trityl, benzyl, 2,4 dichlorobenzyl andbenzyloxycarbonyl. The preferred protecting group is benzyl; or R ishydrogen which means there is no protecting group on the phenolichydroxy function;

R is a protecting group for the alcoholic hydroxyl group of serine andis selected from the class consisting of those members defined by R or Ris hydrogen which means there is no protecting group on the alcoholicoxygen atom. Preferably R is benzyl;

R is preferably hydrogen or an a-amino protecting group. The a-aminoprotecting group contemplated by R are those known to be useful in theart in the stepwise synthesis of polypeptides. Among the classes ofa-amino protecting groups covered by R are (l) acyl type protectinggroups illustrated by the following: formyl, trifluoroacetyl, phthalyl,toluenesulfonyl (tosyl), benzensulfonyl, nitrophenylsulfenyl,tritylsulfenyl, onitrophenoxy acetyl, chloroacetyl, acetyl,y-chlorobutyryl, etc.; (2) aromatic urethan type protecting groupsillustrated by benzyloxycarbonyl and substituted benzyloxycarbonyl suchas p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphaticurethan protecting groups illustrated [by tert-butyloxycarbonyl,diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl: (4) cycloalkyl urethan type protecting groupsillustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl,cyclohexyloxycarbonyl; (5) thio urethan type protecting groups such asphenylthiocarbonyl; (6) alkyl type protecting groups as illustrated bytriphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such astrimethylsilane. The preferred a-amino protecting group defined by R areselected from the class consisting of tertbutyloxycarbonyl,cyclopentyloxycarbonyl, tertamyloxycarbonyl and d-isobornyloxycarbonyl.

In formula II at least one of RR or R is a protecting group.

A further aspect of the present invention relates to intermediateslinked to a solid resin support. These intermediates are represented bythe formula:

(III) wherein:

R, R and R have the same meaning as in Formula II;

R is selected from the class consisting of R -p-Glu, an a-aminoprotecting group of hydrogen (deprotected oz-amino group). The a-aminoprotecting group is preferably tert-butyloxycarbonyl (tert-Boc) whichprotecting group is also preferably used for protecting the a-aminogroup of all amino acids added in the stepwise solid phase synthesis.However, other a-amino protecting groups may be used such aso-nitrophenylsulfenyl, t-amyloxycarbonyl andbiphenylisopropyloxycarbonyl;

A is an anchoring bond used in solid phase synthesis linked to a solidresin support. A is selected from the class consisting of:

t I resin N c Q support and (Illa) resin o support The symbol d) meansphenyl.".

In selecting a particular side chain protecting group to be used in thesynthesis of the peptides of formula (I), the following rules should befollowed: (a) the protecting group must be stable to the reagent andunder the reaction conditions selected for removing the a-aminoprotecting group at each step of the synthesis, (b) the protecting groupmust retain its protecting properties (i.e. not be split off undercoupling conditions), and (c) the side chain protecting group must beremovable upon the completion of the synthesis containing the desiredamino acid sequence under reaction conditions that will not alter thepeptide chain.

Best Available ear illustrative of pharmaceutally acceptable non-toxicsalts of formula 1 are hydrochloride, hydrolnomide, sulfate, phosphate,maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, andthe lilac.

The peptides of formula (1) are prepared using solid phase synthesis.The synthesis is commenced from the C-terminal end of the peptide usingan a-amino protected resin. Such a starting material can be prepared byattaching an a-amino protecting glycine to a henzyhydrilamine resin, achloromethylated resin or a hy droxymethyl resin, the former beingpreferred. The preparation of benzhydrilamine resin is described by P.Rivaille et al., Helv. 54, 2772 1971) and the prepara tion of thehydroxymethyl resin is described by Bo danszky et 211., Chem. 1nd(London) 38, 159798 (1966). A chloromethylated resin is commericallyavailable from Bio Rad Laboratories Richmond, California. In using thebenzhydrilamine resin an amide anchoring bond is formed with the a-aminoprotected gly- This permits the C-terminal amide function to be obtaineddirectly after the amino acid sequence in the synthesis is complete bycleaving off the resin support to form the glycine amide at theC-terminal portion of the desired peptide of formula (I). When the otherresins are used, the anchoring bond is the benzylester group as definedsupra in Formula (lllb), which after cleavage of the peptide from theresin support must be converted to the Cterminal amide. The preferredprocedure is to ammonalyse the protected peptide off the resin and thenremove the protecting group by hydrogenolysis or by hydrogen fluoridecleavage. An alternate procedure would be to cleave bytransesterification with methanol/(Et) l l and then convert theresulting ester into an amide and subsequently deprotect as describedabove. See J. M. Stewart Solid Phase Peptide Synthesis, pg. 4246 (W. H.Freeman and Co. 1969).

The a-amino protected glycine is coupled to the benzhydrilamine resinwith the aid of a carboxyl group activating compound such asdicyclohexylcarbodiimide. Following the coupling of the or-aminoprotected glycine to the resin support, the cc-amino protecting group isremoved such as by using tritluoracetic acid in methylene chloride,trifluoroacetic acid alone or HCl in dioxane. The deprotection iscarried out at a temperature between about 0C and room temperature.Other standard cleaving reagents and conditions for removal of specificot-amino protecting groups may be used as described in Schroder andLubke, The Peptides, 1 72-75 (Academic Press 1965). After removal of thea-amino protecting group the remaining a-amino protected amino acids arecoupled step-wise in the desired order to obtain a compound of formula(1). However, as an alternate to adding each amino acid separately tothe reaction, some of them may be coupled prior to addition to the solidphase reactor. 1f the C-terminal end of the peptide unit is representedby glycine or proline and the coupling is carried out with a minimum ofracemization is encountered with proline and no problems are encounteredwith glycine which has no asyrnctric centre. Each protected amino acidor amino acid sequence, is introduced into the solid phase reactor inabout a four-fold excess and the coupling is carried out in a medium ofdimethylformamide: methylene chloride (1:1) or in dimethylformamide ormethylene chloride alone. In cases where in complete coupling occurredthe coupling procedure is repeated before removal of the ct-aminoprotecting group, prior to the coupling of the next amino acid to thesolid phase reactor. The success of the coupling reaction at each stageof the synthesis is monitored by the ninhydrin reaction as described byE. Kaiser et al., Analyt. Biochem, 34, 595 1970).

After the desired amino acid sequence has been synthesized, the peptideis removed from the resin support by treatment with a reagent such ashydrogen fluoride which not only cleaves the peptide from the resin butalso cleaves all remaining side chain protecting groups and the a-aminoprotecting group (if present) on pyroglutamic acid to obtain directly acompound of formula I in the case where the benzhydrilamine resin wasused. Where a chloromethylated resin is used the peptide may beseparated from the resin by methanolysis after which the recoveredproduct is chromatographed on silica gel and the collected fractionsubject to ammonalysis to convert the methyl ester to the C-terminalamide. Any size chain protecting group may then be cleaved as previouslydescribed or by other procedures such as catalytic reduction (cg. Pd onC) using conditions which will keep the Trp moiety intact. When usinghydrogen fluoride for cleaving, anisole is included in the reactionvessel to prevent the oxidation of labile amino acid (eg tryptophan).

The solid phase synthesis procedure discussed supra is well know in theart and has been essentially de scribed by M. Monahan et al., C. R.Acad. Sci, Paris, 273 508 (1971).

The nomenclature used for peptides is described by Schroder and Lubke,supra, pp viiixxix and in Biochemistry 11, 1726-1732 (1972).

The following examples are illustrative of the preparation of thecompounds formulas I through 111.

EXAMPLE 1 L-Pyroglutamyl-D-Phenylalanyl-L-tryptophyl-O-benzyl-L-seryl-O-benzyl-L-tyrosyl-D-leucyl-Lleucyl- N -N0 L-arginyhL-prolylglycyl benzhydrilamine resin Benzhydryiamine resin (20.0 g.) isput in a Merrifield vessel of 300 ml. capacity and put through thefollow ing wash cycle: a) methylene chloride; 1)) 1:1 trifluoroaceticacid-methylene chloride (3 times for 10 minutes each); c) methylenechloride; (1) methanol; c) 12.5% triethylamine in dimethylforrnamide(two times for 10 minutes each); f) methanol (two times); g) methylenechloride (two times), allowing a contact time of at least 3 minuteseach, if not indicated otherwise.

The resin so prepared is then gently shaken with tbntyloxycarbonylglycine (3.65 g, 21 mrnole) in 1:1 methylene chloridedimethylformamideand 25.6 ml. of 1M dicyclohexylcarbodiimide in methylene chloride isadded in three portions over a period of 30 minutes. Shaking iscontinued at ambient temperature for a total of 18 hours. Thepeptideresin is then washed succes sively with methanol, methylenechloride, methanol (twice), and methylene chloride (twice). To test forcompleteness of reaction, the peptide-resin is subjected to a ninhydrintest following the procedure of E. Kaiser et al., AnalyticalBiochemistry 34, 595 (1970).

The deprotection of the attached amino acid is carried out as follows:The peptide-resin is treated with a 1:1 solution of trifluoroaceticacid-methylene chloride (three times for minutes each), then step (0)through (g), as described above for the wash cycle are performed. Again,a sample of the peptide-resin is subjected to a ninhydrin test to checkfor completeness of reaction. The sample is now strongly positiveindicating deprotection of the glycine molecule attached to the resin.

The following amino acid residues are then introduced consecutively:t-Boc-L-proline (21 mmoles, 25.6 mmoles DCC), t-Boc-nitro-L-arginine (21mmoles, 25.6 mmoles DCC), t-Boc-L-leucine (21 mmoles, 25.6 mmoles DCC).Each coupling step is carried out in a medium of methylenechloride-dimethylformamide (1:1) and the removal of the a-aminoprotecting group at each step is performed as described for thedeprotection of the t-Boc-glycine-resin.

At this point, the washed tetrapeptide is dried and the synthesiscontinued with 6 g. of the tetrapeptide-resin. The next amino acid addedis t-Boc-D-leucine (6.8 mmoles, 8.5 mmoles DCC), followed bydeprotection then t-Boc-O-benzyl-L-tyrosine (6.8 mmoles, 8.5 mmoles DCC)is added followed by deprotection, then 't-Boc-O-benzyl-L-serine (6.8mmoles, 8.5 mmoles EXAMPLE 2Pyroglutamyl-D-phenylalanyl-Ltryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-Larginyl-L- prolylglycinamide Removal of theprotecting groups and cleavage of the decapeptide from the resin (2.5g.) is accomplished by treating the dried peptide-resin of Example 1 invacuo with liquid hydrogen fluoride ml.) and anisole (10 ml.) at icebath temperature for one hour. The hydrogen fluoride is removed byvacuum distillation and the anisole removed by washing with ether. Thepeptide is dissolved in 10% acetic acid and removed from the resin byfiltration. Lyophilization yields the desired decapeptide as a whitefluffy powder.

EXAMPLE 3 Purification and characterization ofPyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-prolylglycinamide acetate The crudepeptide is dissolved in a minimum volume of 0.2N acetic acid, applied toa Bio-Gel P-2 200400 mesh gel filtration column (2.6 cm X cm) and elutedwith the same solvent. Fractions of 9 ml. each are collected. Thefractions (3240) containing the desired peptide are located by Paulyspot test and UV analysis. After pooling and lyophilization, a whitefluffy powder (460 mg.) is obtained.

A partition column of Sephadex G-25 fine (2.6 cm X 90 cm is prepared byequilibration with lower phase and then upper phase of the BAW solventsystem (nbutanol:acetic acid:water, 4:1:5, V ==l65 ml.).

The lyophilized peptide from above is applied in a minimum volume ofupper phase. Elution with upper phase (6 ml. fractions) affords thedesired product which is located as described above. After pooling andlyophilization, a white fluffy powder (237 mg.) is obtained.

The optical rotation is measured on a Carl Zeiss LEP A2 photoelectricprecision polarimeter, [M 26.0 (c=0.795, 1% acetic acid); Amino acidanalysis gives the following ratios: Ser (0.97), Glu (1.03), Pro (0.9),Gly (1.0), Leu plus D-Leu (2.0), Tyr (1.2), D-Phe (1.0), Trp (0.73), Arg1.0); R;(on silica): 0.34 (n-butanolzacetic acid:water 4: l :5

The peptide (20 pg load) is homogeneous in three TLC systems (acidic,neutral, and basic) when examined under ultraviolet light, iodine vapor,and Pauly reagent.

The compounds of formula I possess anti-ovulatory activity and hence arepotentially useful in inhibiting fertility in female mammals. In testsconducted with female rats (225 to 250 grams body weight) ovulationinhibition was achieved at a dose of about 24 mg/kg. The test wasconducted with mature Sprague-Dawley rats, normally cycling,unanesthetized, proestrous rats. On the afternoon of proestrous, eachrat in the test group received six subcutaneous injections of theacetate salt of formula I in corn oil, each injection being given a halfhour following the previous injection. The rats are sacrificed the nextmorning and the number of animals ovulating and the number of ova shedare recorded following the procedure described by E. S. France,Neuroendocrinology 6, pp 77-89 (1970). The absence of or a significantdecrease in the number of ova is the criterion for an anti-ovulationeffect. At a dose of 1 mg per injection inhibition of ovulation wasachieved.

The compounds of formula 1 can be administered to mammals intravenously,subcutaneously, intramuscularly or orally for fertility inhibition andcontrol. The effective dosage will vary with the form of administrationand the particular species of mammal to be treated. A typical dosage isa physiological saline solution containing a compound of formula 1administered in a dose range of between about 20 to 30 mg/kg of bodyweight. Oral administration may be in either solid or liquid form.

What is claimed is:

1. A compound selected from the group consisting of and its non-toxicsalts; wherein R is selected from the class consisting of NH OH,O-(lower)-alkyl and O-benzyl; R is a protecting 7 group for the N N" andN nitrogen atoms of arginine selected from the class consisting ofnitro, tosyl, benzyloxycarbonyl, and adamantyloxycarbonyl; or R ishydrogen;

R is a protecting group for the phenolic hydroxyl group of tyrosineselected from the class consisting of acetyl, tosyl, benzoyl,tert-butyl, tetrahydropyranyl, trityl, benzyl, 2,4 dichlorobenzyl andbenzyloxycarbonyl or R is hydrogen;

R is a protecting group for the alcoholic hydroxyl group of serine andis selected from the class consisting of acetyl, tosyl, benzoyl,tetrahydropyranyl, tert-butyl, trityl, 2,4 dichlorobenzyl, benzyl andbenzyloxycarbonyl; or R is hydrogen;

R is selected from the class consisting of hydrogen or an a-aminoprotection group, with the proviso that at least one of R, R and R is aprotecting group.

2. A compound according to claim 1 wherein R is 3. A compound accordingto claim 1 wherein R is NH R is nitro, R is benzyl, R is benzyl and R ishydrogen.

4. A compound according to claim 1 which is selected from:L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl-glycinamide and its non-toxic acid addition salts.

5. A compound of the formula: i

wherein:

R, R, R and R have the same meaning as in claim 1; R is selected fromthe class consisting of R-p- Glu-, an a-amino protecting group orhydrogen; and A is selected from the class consisting of H H l l N c 9and resin I support O -CH nitro and R and R are benzyl.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OFL-P-GLU-D-PHE-L-TRP-L-SER-L-TYR-D-LEU-L-LEU-L-ARG-LPRO-GLY-NH2 ANDR4-L-P-GLU-D-PHE-L-TRP-L-SER(R3)-L-TYR-(R2)-D-LEU-LLEU-L-ARG-(NG-R1)-L-PRO-GLY-R AND ITS NON-TOXIC SALTS, WHEREIN R IS SELECTED FROM THE CLASSCONSISTING OF NH2, OH, O(LOWER)-ALKYL AND O-BENZYL, R1 IS A PROTECTINGGROUP FOR THE N$, NW AND NW'' NITROGEN ATOMS OF ARGININE SELECTED FROMTHE CLASS CONSISTING OF NITRO, TOSYL, BENZYLOXYCARBONYL, ANDADAMANTYLOXYCARBONYL, OR R1 IS HYDROGEN, R2 IS A PROTECTING GROUP FORTHE PHENOLIC HYDROXYL GROUP OF TYROSINE SELECTED FROM THE CLASSCONSISTING OF ACETYL, TOSYL, BENZOYL, TERT-BUTYL, TETRAHYDROPYRANYL,TRITYL, BENZYL, 2,4 DICHLOROBENZYL AND BENZYLOXYCARBONYL OR R2 ISHYDROGEN, R3 IS A PROTECTING GROUP FOR THE ALCOHOLIC HYDROXYL GROUP OFSERINE AND IS SELECTED FROM THE CLASS CONSISTING OF ACETYL, TOSYL,BENZOYL, TETRAHYDROPYRANYL, TERT-BUTYL, TRITYL, 2,4 DICHLOROBENZYL,BENZYL AND BENZYLOXYCARBONYL, OR R3 IS HYDROGEN, R4 IS SELECTED FROM THECLASS CONSISTING OF HYDROGEN OR AN A-AMINO PROTECTION GROUP, WITH THEPROVISO THAT AT LEAST ONE OF R1, R2 AND R3 IS A PROTECTING GROUP.
 2. Acompound according to claim 1 wherein R is NH2.
 3. A compound accordingto claim 1 wherein R is NH2, R1 is nitro, R2 is benzyl, R3 is benzyl andR4 is hydrogen.
 4. A compound according to claim 1 which is selectedfrom:L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl-glycinamide and its non-toxic acid additionsalts.
 5. A compound of the formula:R5-D-Phe-L-Trp-Ser(R3)-L-Tyr(R2)-D-Leu-L-Leu-L-Arc(NG-R1)-L-Pro-Gly-Awherein: R1, R2, R4 and R3 have the same meaning as in claim 1; R5 isselected from the class consisting of R4-p-Glu-, an Alpha -aminoprotecting group or hydrogen; and A is selected from the classconsisting of
 6. A compound according to claim 5 wherein R5 is an Alpha-amino protecting group which is selected from the class consisting oftert-butyloxycarbonyl, o-nitrophenylsulfenyl, tert-amyloxycarbonyl andbiphenylisopropyloxycarbonyl.
 7. A compound according to claim 5 whereinA is a benzhydrilamine resin and R5 is R4-p-Glu and R4 is hydrogen.
 8. Acompound according to claim 7 wherein R1 is nitro and R2 and R3 arebenzyl.